Project Summary/Abstract Abilita Bio, an innovation-driven biotechnology company, is seeking SBIR Phase I funding for the discovery of novel therapeutic antibodies targeting the human voltage-gated sodium (Nav) channels Nav1.7 and Nav1.8, for the non-opioid treatment of pain. Nearly 50 million adults in the US suffer from chronic pain, which results an estimated combined burden that exceeds $600 billion annually and eclipses the combined economic costs of diabetes, heart disease and cancer. The prevalence of chronic pain lies at the root of an ongoing epidemic of prescription painkiller abuse. Current pain management strategies rely heavily on the prescription of opioids as oxycodone and hydrocodone, which are effective, but are associated with an intrinsic risk of addiction and potentially fatal side effects like respiratory depression. Select members of the human Nav channel family are critically involved in the sensation of pain and are expressed almost exclusively in the peripheral nervous system. The discovery and development of specific Nav channel antagonists is considered to be a strategy with great potential for treatment of chronic pain and may have further applications to neuropathic and cancer pain. Despite the importance of subtype selectivity to both efficacy and safety, current Nav channel-targeting drugs are poorly selective among the subtypes. To improve overall clinical efficacy and remove the potentially devastating side effects of Nav- targeting drugs such as cardiac toxicity, we propose to develop subtype-specific antibody-based therapeutics. To date, the challenges associated with the discovery of antibodies targeting ion channels are insurmountable. The main reasons for the lack of therapeutic antibodies or other ?antibody-like? agents targeting Nav channels are their very low expression, membrane extractability and stability when removed from their natural membrane environment. Abilita Bio?s directed evolution-based discovery engine offers an innovative solution to this difficulty through the generation of Nav variants representing structurally and functionally enhanced versions of natural channels with unprecedented expression levels and improved thermostability. We call these variants Enabled Membrane Proteins (EMPs?). The availability of highly expressing stable and functionally folded EMPs? will enable the isolation of novel therapeutic antibodies using a combination of in vivo and in vitro antibody discovery methods as described more in detail in the proposal. The use of therapeutic antibody fragments to target human Nav channel family would have a tremendous potential for the treatment of pain because of their typical high affinity, exquisite specificity and low toxicity relative to small-molecule therapeutics. Nav channel antibody or ?antibody-like? antagonists can possibly represent a strategy for the safe treatment of chronic pain and may have further applications to neuropathic and cancer pain. Funds are requested to (a) generate additional mutational gene libraries for proposed targets Nav1.7 and Nav1.8, and select for enhanced EMP? variants using our directed evolution discovery engine; (b) characterize the selected EMPs? variants for expression, function and enhanced stability when produced from mammalian cells and confirm their relevant structure/folding using tool compound binding; (c) select the best candidate Nav1.7 or Nav1.8-EMP?, produce it in large-scale and use it for single domain antibody discovery (nanobody) using a phage-display platform; (d) lastly, the discovered monovalent or dimerized antibody hits will be tested for relevant pharmacology using whole-cell electrophysiology studies (patch-clamp) in order to determine the functional inhibition of Nav channel activity.